Valeria Fiorelli

The Presence of Anti-Tat Antibodies Is Predictive of Long-Term Nonprogression to AIDS or Severe Immunodeficiency: Findings in a Cohort of HIV-1 Seroconverters

The human immunodeficiency virus (HIV) type 1 Tat protein plays a key role in the life cycle of the virus and in pathogenesis and is highly conserved among HIV subtypes. On the basis of this and of safety, immunogenicity, and efficacy findings in monkeys, Tat is being tested as a vaccine in phase 1 trials. Here, we evaluated the incidence and risk of progression to advanced HIV disease by anti-Tat serostatus in a cohort of 252 HIV-1 seroconverters. The risk of progression was lower in the anti-Tat-positive subjects than in the anti-Tat-negative subjects. Progression was faster in the persistently anti-Tat-negative subjects than in the transiently anti-Tat-positive subjects, and no progression was observed in the persistently anti-Tat-positive subjects.

Efficient mucosal delivery of the HIV‐1 Tat protein using the synthetic lipopeptide MALP‐2 as adjuvant

A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell‐mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV‐1 Tat protein as antigen and the synthetic lipopeptide, macrophage‐activating lipopeptide‐2 (MALP‐2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti‐Tat antibody responses, and Tat‐specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freunds adjuvant. Major linear B cell epitopes mapped within aa 1–20 and 46–60, whereas T cell epitopes were identified within aa 36–50 and 56–70. These epitopes have also been described in vaccinated primates and in HIV‐1‐infected individuals with better prognosis. Analysis of the anti‐Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP‐2, as opposed to the Th2 response observed with Tat plus incomplete Freunds adjuvant. Tat‐specific IFN‐γ‐producing cells were significantly increased only in response to Tat plus MALP‐2. These data suggest that Malp‐2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.

HIV-1 Tat addresses dendritic cells to induce a predominant Th1-type adaptive immune response that appears prevalent in the asymptomatic stage of infection

Tat is an early regulatory protein that plays a major role in human HIV-1 replication and AIDS pathogenesis, and therefore, it represents a key target for the host immune response. In natural infection, however, Abs against Tat are produced only by a small fraction (∼20%) of asymptomatic individuals and are rarely seen in progressors, suggesting that Tat may possess properties diverting the adaptive immunity from generating humoral responses. Here we show that a Th1-type T cell response against Tat is predominant over a Th2-type B cell response in natural HIV-1 infection. This is likely due to the capability of Tat to selectively target and very efficiently enter CD1a-expressing monocyte-derived dendritic cells (MDDC), which represent a primary target for the recognition and response to virus Ag. Upon cellular uptake, Tat induces MDDC maturation and Th1-associated cytokines and β-chemokines production and polarizes the immune response in vitro to the Th1 pattern through the transcriptional activation of TNF-α gene expression. This requires the full conservation of Tat transactivation activity since neither MDDC maturation nor TNF-α production are found with either an oxidized Tat, which does not enter MDDC, or with a Tat protein mutated in the cysteine-rich region (cys22 Tat), which enters MDDC as the wild-type Tat but is transactivation silent. Consistently with these data, inoculation of monkeys with the native wild-type Tat induced a predominant Th1 response, whereas cys22 Tat generated mostly Th2 responses, therefore providing evidence that Tat induces a predominant Th1 polarized adaptive immune response in the host.

Phase I therapeutic trial of the HIV-1 Tat protein and long term follow-up

A randomized, double blind, placebo-controlled phase I vaccine trial based on the native Tat protein was conducted in HIV-infected asymptomatic individuals. The vaccine was administered five times subcute with alum or intradermally without adjuvant at 7.5microg, 15microg or 30microg doses, respectively. The Tat vaccine was well tolerated both locally and systemically and induced and/or maintained Tat-specific T helper (Th)-1 T-cell responses and Th-2 responses in all subjects with a wide spectrum of functional anti-Tat antibodies, rarely seen in HIV-infected subjects. The data indicate the achievement of both the primary (safety) and secondary (immunogenicity) endpoints of the study.

The preventive phase I trial with the HIV-1 Tat-based vaccine.

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials based on its role in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune response with the asymptomatic stage as well as on its sequence conservation among HIV clades. A randomized, double blind, placebo-controlled phase I study (ISS P-001) was conducted in healthy adult volunteers without identifiable risk of HIV infection. Tat was administered 5 times monthly, subcute in alum or intradermic alone at 7.5 microg, 15 microg or 30 microg, respectively (ClinicalTrials.gov identifier: NCT00529698). Vaccination with Tat resulted to be safe and well tolerated (primary endpoint) both locally and systemically. In addition, Tat induced both Th1 and Th2 type specific immune responses in all subjects (secondary endpoint) with a wide spectrum of functional antibodies that are rarely seen in natural infection, providing key information for further clinical development of the Tat vaccine candidate.

The therapeutic phase I trial of the recombinant native HIV-1 Tat protein.

The native HIV-1 Tat protein was chosen as a vaccine candidate based on its key role in the virus life cycle and on the correlation of Tat-specific immune responses with the asymptomatic stage and lower disease progression rates, but also due to its sequence conservation amongst the various HIV clades as well as the adjuvant effects on dendritic cells. Safety, immunogenicity and efficacy data in monkeys support the development of this vaccine concept.

Parallel conduction of the phase I preventive and therapeutic trials based on the Tat vaccine candidate

The native HIV-1 Tat protein was chosen as vaccine candidate for phase I clinical trials in both uninfected (ClinicalTrials.gov identifier: NCT00529698) and infected volunteers (ClinicalTrials.gov identifier: NCT00505401). The rationale was based on the role of Tat in the natural infection and AIDS pathogenesis, on the association of Tat-specific immune responses with the asymptomatic stage and slow-progression rate as well as on its sequence conservation among HIV clades (http://www.hiv1tat-vaccines.info/). The parallel conduction in the same clinical centers of randomized, double blind, placebo-controlled phase I studies both in healthy, immunologically competent adults and in HIV-infected, clinically asymptomatic, individuals represents a unique occasion to compare the vaccine-induced immune response in both the preventive and therapeutic setting. In both studies, the same lot of the native Tat protein was administered 5 times, every four weeks, subcute (SC) with alum adjuvant or intradermic (ID), in the absence of adjuvant, at 7.5 microg, 15 microg or 30 microg doses, respectively. The primary and secondary endpoints of these studies were the safety and immunogenicity of the vaccine candidate, respectively. The study lasted 52 weeks and monitoring was conducted for on additional 3 years. The results of both studies indicated that the Tat vaccine is safe and well tolerated both locally and systemically and it is highly immunogenic at all the dosages and by both routes of administration. Vaccination with Tat induced a balanced immune response in uninfected and infected individuals. In particular, therapeutic immunization induced functional antibodies and partially reverted the marked Th1 polarization of anti-Tat immunity seen in natural infection, and elicited a more balanced Th1/Th2 immune response. Further, the number of CD4 T cells correlated positively with anti-Tat antibody titers. Based on these results, a phase II study is ongoing in infected drug-treated individuals (http://www.hiv1tat-vaccines.info/).

Red blood cell-mediated delivery of recombinant HIV-1 Tat protein in mice induces anti-Tat neutralizing antibodies and CTL

The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model. HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs). The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin-avidin-biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat.Balb/c mice were then immunized with 10 microg of soluble Tat in complete Freunds adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5 microg soluble Tat in incomplete Freunds adjuvant or with 20 ng of RBC-coupled Tat, respectively. Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies. In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat. These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies.

Mucosal delivery of the human immunodeficiency virus-1 Tat protein in mice elicits systemic neutralizing antibodies, cytotoxic T lymphocytes and mucosal IgA

Human immunodeficiency virus (HIV)-1 Tat protein induces protection in non-human primates upon systemic vaccination. In view of the design of mucosal vaccines against HIV-1 we studied the immune response to native Tat (aa 1-86) in mice following intranasal delivery of the protein with two mucosal adjuvants, Escherichia coli heat-labile enterotoxin (LT) and LT-R72, a non-toxic mutant of LT. Immunization with Tat and the two adjuvants induced in BALB/c but not in C57BL/6 mice high and persistent levels of serum IgG and secretory IgA in vaginal and intestinal fluids. Mice sera neutralized Tat and recognized two epitopes mapping in the regions 1-20 and 46-60. Furthermore, their splenocytes proliferated and secreted IFN-gamma and IL-6 in response to Tat. Finally, CTLs were also elicited and they recognized an epitope localized within aa 11-40 of Tat.